CN107190005B - Applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment - Google Patents
Applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment Download PDFInfo
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
The invention discloses applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment, the lncRNA is LOC101930114, and LOC101930114 is in low expression in pulmonary adenocarcinoma.Further, the expression present invention finds promotion LOC101930114 genes can promote the apoptosis of lung adenocarcinoma cell, suppress the migration and invasion and attack of cancer cell.The present invention provides new clinical means for the diagnose and treat of adenocarcinoma of lung.
Description
Technical field
The invention belongs to biomedicine field, is related to applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment,
The lncRNA biomarkers are LOC101930114.
Background technology
The death rate of lung cancer worldwide occupy the first.At present in China, the incidence of lung cancer is in what is risen year by year
Trend.Lung cancer can be divided into Small Cell Lung Cancer and non-small cell lung cancer (non-small cell according to the difference of clinicopathologic pattern
Lung cancer, NSCLC) two classes.Wherein, non-small cell lung cancer is most common in lung cancer, accounts for 85% in lung cancer, lung gland
Cancer is wherein most important part again.For non-small cell lung cancer, operative treatment is still the first choice of clinical treatment, for morning
The Patients with Non-small-cell Lung (I and II phases, and comparatively ideal IIIA phases patient) of phase, radical resection of pulmonary carcinoma is still primary treatment
Method.Pertinent literature reports that 1,2,5 years postoperative survival rates of IIIA phase patients are apparently higher than non-operative treatment group.For losing
For the patients with terminal on lung cancer radical operation opportunity, treatment should be based on chemotherapy.In recent years, as accurate radiotherapy technology is sent out
The effect of exhibition, by reducing radiotherapy number, increases the dosage of single radiotherapy, radiotherapy is greatly improved.
With the mankind to tumour gene and molecular level deep understanding, molecular targeted agents due to its accuracy and
Relatively low poisonous side effect of medicine, is increasingly subject to the attention and concern of medical profession personage, becomes the heat of current lung cancer research field
Point.As at present in non-small cell therapy field TKT (small molecule tyrosine kinase inhibitors) the most ripe.Such medicine passes through
It is combined with the Intracellular phosphorylation enzyme effect site of EGFR, suppresses the activation of phosphorylase, so as to suppresses EGFR and downstream letter
The activation of number path.The research emphasis of current molecular targeted therapy is mainly to find more efficient cancer coding and controlling gene,
By the research to its mechanism of action, and then the expression of gene is adjusted and controlled, suppress the development of tumour.
RNA can be divided into mRNA (mRNA), transfer RNA (tRNA) and rRNA according to classical taxonomy method.Except this
Outside, according to whether can have the function of that encoding proteins can be divided into coding RNA and non-coding RNA (non-coding RNA).It is early
Phase, research of the people for RNA is only limitted to coding RNA, but is found by research, and part RNA only accounts for the 1% of genome, remains
Under be non-coding RNA.Then, the excavation for non-coding RNA function becomes the weight of scientists concern in recent years
Point.And ncRNA according to contained base quantity number can be divided into small molecule non-coding RNA (miRNA, microRNA) and long
Chain non-coding RNA (lncRNA, long non-coding RNA).Wherein, miRNA has been achieved for by research these years
Significant progress, the new drug that some miRNA have become neoplasm targeted therapy apply to clinic.But the research of lncRNA is firm at present
In the starting stage, in this field, also very big blank needs researcher to go to fill up.
In these years, in the various tumours of the mankind, the lncRNA for constantly having new unconventionality expression is mined.Ground from current
From the point of view of studying carefully, during effects of the lncRNA in tumour runs through the whole occurrence and development of tumour.Both can be to the week of cancer cell
Phase, apoptosis, transfer etc. have an impact, also can in the aspect of epigenetic macro adjustments and controls tumour cell many features.Due to
The occurrence and development of lncRNA and tumour have important relationship, it is seen that it may have in the diagnosis and treatment of tumour it is great latent
Power.In terms of diagnosis, often there is the unconventionality expression of lncRNA in tumor tissues, it is likely to become important tumor markers, especially
It is early stage some tumours, and due to tumor tissues very little, traditional Imaging Method is difficult to find.And by detecting in blood
Certain lncRNA it is horizontal, the possibility that patient suffer from certain tumour can be but understood with fast and easy.In terms for the treatment of, exist to lncRNA
Effect and the research of mechanism can find new cancer target in cancer, develop new efficient, accurate, less toxic side effect
Anti-cancer drugs.
At present, the research of lncRNA is still in infancy, to the understanding of its Mechanism and FunctionsDNA yet among exploration.
LncRNA not only plays various important biological functions in organism, while also assists in the occurrence and development of a variety of diseases.
In tumor research field, lncRNA is still a relatively unknown field, it would be desirable to by the continuous research to lncRNA come
New opportunity is provided for the diagnosis and treatment of tumour.
The content of the invention
In order to make up for the deficiencies of the prior art, it is an object of the invention to provide a kind of available for adenocarcinoma of lung diagnosis and treatment
LncRNA biomarkers, lncRNA biomarkers provided by the present invention for human lung adenocarcinoma have higher sensitivity and
Specificity, can be used for the detection of adenocarcinoma of lung as new biomarkers.
To achieve these goals, the present invention adopts the following technical scheme that：
The present invention provides a kind of biomarker of adenocarcinoma of lung, the marker is LOC101930114, its nucleotide
Sequence is as shown in SEQ ID NO.1.
The present invention provides above-mentioned LOC101930114 genes and its expression product to prepare the product of diagnosis adenocarcinoma of lung
In application.
Further, product recited above can by detect the expression of the LOC101930114 genes in sample come
Whether diagnosis patient suffers from adenocarcinoma of lung, and LOC101930114 expresses downward in patients with lung adenocarcinoma.
Further, the product of the diagnosis adenocarcinoma of lung includes chip, preparation or kit.
The present invention provides a kind of chip, preparation or kit, it contains above-mentioned LOC101930114.
Further, the chip can be used for multiple genes of the detection including LOC101930114 genes (for example, and lung
The relevant multiple genes of gland cancer) expression；The kit can be used for detection including LOC101930114 genes
The expression of multiple genes (for example, with the relevant multiple genes of adenocarcinoma of lung).
The present invention provides chip recited above, preparation or kit answering in the product for preparing diagnosis adenocarcinoma of lung
With.
The present invention provides purposes of the above-mentioned LOC101930114 in human lung adenocarcinoma diagnosis and treatment medicine is screened.
The present invention provides applications of the LOC101930114 in the pharmaceutical composition for preparing prevention or treatment adenocarcinoma of lung.
Further, described pharmaceutical composition includes LOC101930114 genes and/or the accelerating agent of its expression product.
The present invention provides a kind of pharmaceutical composition for being used to preventing or treating adenocarcinoma of lung, described pharmaceutical composition includes controlling
Treat a effective amount of accelerating agent recited above.
Further, described pharmaceutical composition further includes and other medicine classes of the accelerating agent compatibility and pharmaceutically acceptable
Carrier and/or auxiliary material.
The present invention medicine can also with the drug combination of other treatment adenocarcinoma of lung, other therapeutic compound can with it is main
Active ingredient be administered simultaneously, or even be administered simultaneously in same composition.
Other treatments can also be individually given with single composition or the dosage form different from main active ingredient
Property compound.The Fractional of main component can be administered simultaneously with other therapeutic compounds, and other dosage can be independent
Administration.Over the course for the treatment of, can be according to the order of severity, the frequency of recurrence and the physiologic response of therapeutic scheme of symptom, adjustment
The dosage of pharmaceutical composition of the present invention.
The advantages of the present invention：
Present invention firstly discovers that the differential expression of LOC101930114 is to the occurrence and development of adenocarcinoma of lung related, pass through detection
The expression of LOC101930114 in subject's sample, it can be determined that whether subject suffers from adenocarcinoma of lung, so as to instruct clinician
Prevention scheme or therapeutic scheme are provided to subject.
Present invention finds a kind of new biomarker-LOC101930114 of adenocarcinoma of lung, using biomarker come
Prevention or treatment adenocarcinoma of lung, it is more sensitive, special.
Brief description of the drawings
Fig. 1 is the expression figure in pulmonary adenocarcinoma using QPCR detection LOC101930114 genes；
Fig. 2 is the transfected condition figure in lung adenocarcinoma cell using QPCR detections LOC101930114；
Fig. 3 is the influence figure bred with CCK-8 methods detection LOC101930114 gene pairs lung adenocarcinoma cell；
Fig. 4 is the influence figure with flow cytomery LOC101930114 gene pairs Apoptosis of Lung Adenocarcinoma Cell；
Fig. 5 is the influence figure for being migrated and being attacked to lung adenocarcinoma cell using Transwell cells detection LOC101930114；
Wherein figure A is the influence figure that LOC101930114 migrates lung adenocarcinoma cell；It is that LOC101930114 invades lung adenocarcinoma cell to scheme B
The influence figure attacked.
Specific embodiment
The present invention, by largely screening, is found that in adenocarcinoma of lung first by in-depth study extensively
Specific low expression is presented in LOC101930114.It is demonstrated experimentally that the expression by specifically improving LOC101930114 is put down or is expressed
The activity of product, can effectively inhibit the growth and invasion and attack of lung adenocarcinoma cell, so as to reach the effect for suppressing adenocarcinoma of lung.
Biomarker
Term " biomarker " is its expression in tissue or cell and normal or healthy cell or tissue
Expression compares any gene to change.
It would be recognized by those skilled in the art that the practicality of the present invention is not limited to the marker gene of the present invention
The gene expression of any specific variants is quantified.As nonrestrictive example, marker gene can have SEQ ID NO.1
The nucleotide sequence specified.In some embodiments, it has and at least 85% the same or similar cDNA of listed sequence
Such as above-mentioned listed sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% of sequence or at least
99% the same or similar cDNA sequence.
The present invention can utilize any method known in the art measure gene expression.Those skilled in the art should manage
Solution, the means for measuring gene expression are not the importances of the present invention.The table of biomarker can be detected on transcriptional level
Up to level.
In some embodiments, the expression of biomarker is detected on transcriptional level.Hybridize skill using nucleic acid
A variety of methods that art carries out specific DNA and RNA measurement are known to the skilled in the art.Certain methods are related to electrophoretic separation
(for example, for detecting the Southern traces of DNA and Northern traces for detecting RNA), but can also be unfavorable
The measurement (for example, passing through Dot blot) of DNA and RNA is carried out in the case of separated with electrophoresis.Genomic DNA is (for example, come from
People) Southern traces can be used for screening restriction fragment length polymorphism (RFLP), polypeptide of the present invention is influenced with detection
The presence of inherited disorder.The RNA of form of ownership can be detected.
Chip
The lncRNA chips of the present invention include：Solid phase carrier；And it is fixed in order on the solid phase carrier
Oligonucleotide probe, the oligonucleotide probe specifically correspond to the part or all of sequence shown in LOC101930114
Row.
Specifically, can lncRNA according to the present invention, design suitable probe, be fixed on solid phase carrier, shape
Into " oligonucleotide arrays "." oligonucleotide arrays " refer to (addressable i.e. with distinctive with addressable point
The position that address is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to
Need, oligonucleotide arrays can be divided into multiple sub- battle arrays.
Term " probe " refers to the molecule that can be combined with the particular sequence or subsequence or other parts of another molecule.It is unless another
Point out, term " probe " is often referred to match by complementary base and another polynucleotides (often referred to as " target polynucleotide ")
With reference to polynucleotide probes.Lack sufficient sequence complementarity according to the stringency of hybridization conditions, probe energy and with the probe
Target polynucleotide combines.Probe can make direct or indirect mark, its scope includes primer.Crossing system, including, but it is unlimited
In：Solution phase, solid phase, mixed phase or in situ hybridization determination method.
Term " complementary " or " complementarity ", which are used to refer to, passes through relevant polynucleotides (that is, the nucleosides of basepairing rule
The sequence of acid).For example, sequence " 5'-A-G-T-3' " is complementary with sequence " 3'-T-C-A-5' ".Complementarity can be " part ",
The base of only few of which nucleic acid is matched according to basepairing rule.Alternatively, can also exist between nucleic acid " complete " or
It is " total " complementary.Complementarity between nucleic acid chains has significant impact for the hybridization efficiency between nucleic acid chains and intensity.
This amplified reaction and rely on nucleic acid between combination detection method in be even more important.
Term " stringency ", which is used to refer to, carries out the residing condition of nucleic acid hybridization：Temperature, ionic strength and other compounds
The presence of (such as organic solvent).Under " low stringency condition ", nucleotide sequence of interest will with its exact complementary sequence, have
The sequence of single base mispairing, closely related sequence (for example, sequence with 90% or more high homology) and only portion
Divide sequence (for example, sequence with the 50-90% homologys) hybridization of homology.It is of interest under " medium stringent conditions "
Nucleotide sequence by only with its exact complementary sequence, have single base mispairing sequence and closely related sequence (for example,
90% or more high homology) hybridization.Under " high stringency condition ", nucleotide sequence of interest general and its exact complementary sequence
The sequence that (condition for depending on such as temperature) has single base mispairing hybridizes.In other words, under the conditions of high stringency,
Temperature can be raised to exclude to hybridize with the sequence with single base mispairing.
Oligonucleotide probe in the present invention for LOC101930114 genes can be that DNA, RNA, DNA-RNA are fitted together to
Body, PNA or other derivatives.The length of the probe does not limit, as long as completing specific hybrid and purpose nucleotide sequence
Specific binding, any length can.The length of the probe can be as short as 25,20,15,13 or 10 bases longs.Equally,
The length of the probe can be grown to 60,80,100,150,300 base-pairs or longer, or even whole gene.Due to different spies
Pin length has hybridization efficiency, signal specificity different influences, and the length of the probe is typically at least 14 base-pairs, most
Length is usually no more than 30 base-pairs, optimal with 15-25 base-pair with the length of purpose nucleotide sequence complementation.The probe
Self-complementary sequences are most preferably less than 4 base-pairs, in order to avoid influence hybridization efficiency.
Heretofore described solid phase carrier can use the various common used materials in genetic chip field, such as, but not limited to nylon
Film, slide or silicon chip, unmodified slide, plastic sheet through active group (such as aldehyde radical, amino) modification etc..
The LOC101930114 chips prepare the common manufacturing method that can use biochip known in the art.
For example, if solid phase carrier, can be by widow containing amido modified poly- dT strings using modification slide or silicon chip, 5 ' ends of probe
Nucleotide probe is configured to solution, then using point sample instrument by its point modification slide or silicon chip on, be arranged in predetermined sequence
Or array, then fixed by standing overnight, so that it may obtain the lncRNA chips of the present invention.
Kit
The present invention provides a kind of kit, the kit can be used for the expression of detection LOC101930114.
Preferably, also containing being useful for the label of labeled RNA sample in the preparation or kit, and with the mark
Remember the corresponding substrate of thing.In addition, it may also include in the kit for extracting needed for RNA, PCR, hybridization, colour developing etc.
Various reagents, include but not limited to：Extract, amplification liquid, hybridization solution, enzyme, comparison liquid, nitrite ion, washing lotion etc..It is in addition, described
Kit in further include operation instructions and/or chip image analysis software.
Drug screening
The present invention provides purposes of the LOC101930114 in human lung adenocarcinoma diagnosis and treatment medicine is screened.I.e.：Use candidate substances
The system of processing expression LOC101930114；With the expression of LOC101930114 or activity in the detection system；If the time
Material is selected to promote expression or the activity of LOC101930114, then it is to suppress the potential material of adenocarcinoma of lung to show the candidate substances.
The system of the expression LOC101930114 for example can be cell (or cell culture) system, and the cell can be
The cell of endogenous expression LOC101930114；Or can be the cell for recombinantly expressing LOC101930114.The expression
The system of LOC101930114 can also be subcellular fraction system, solution system, organizational framework, organ systems or animal system (such as
The animal model of animal model, preferably non-human mammal, such as mouse, rabbit, sheep, monkey) etc..
The accelerating agent and pharmaceutical composition of LOC101930114
Based on the discovery of the present invention, the present invention provides a kind of pharmaceutical composition, described pharmaceutical composition includes
The accelerating agent of LOC101930114.
The accelerating agent of the LOC101930114 refers to any improve LOC101930114 genes or expression product is steady
Qualitative, up-regulation LOC101930114 expression, the effective acting time of increase lncRNA LOC101930114 or promotion
The material of the transcription of LOC101930114 genes, these materials are used equally for the present invention, as raising LOC101930114
Gene expresses useful material, so as to for preventing or treating adenocarcinoma of lung.
As a kind of preferred embodiment of the present invention, the accelerating agent of the LOC101930114 is that one kind contains
The expression vector of LOC101930114.The expression vector usually also contains promoter, replication orgin and/or marker gene
Deng.
Method well-known to those having ordinary skill in the art can be used to build the required expression vector of the present invention.These methods include
Recombinant DNA technology in vi, DNA synthetic technologys, In vivo recombination technology etc..The expression vector preferably includes one or more
Selected marker, to provide the phenotypic character for the host cell for being used to select conversion, such as kalamycin, gentamicin, tide
Mycin, amicillin resistance.
In the present invention, be various carriers known in the art, such as commercially available carrier including plasmid, clay, bacteriophage,
Virus etc..Importing of the expression vector into host cell can use electroporation, calcium phosphate method, liposome method, DEAE Portugals to gather
The known methods such as sugared method, microinjection, viral infection, the combination of liposome transfection and cell-membrane permeable peptide.
Term " host cell " includes prokaryotic and eukaryotic.The example of common prokaryotic host cell includes large intestine
Bacillus, hay bacillus etc..Common eukaryotic host cell includes yeast cells, insect cell and mammalian cell.It is preferred that
The host cell is eukaryotic, such as Chinese hamster ovary celI, COS cells.
Pharmaceutical composition
Pharmaceutical composition in the present invention include LOC101930114 accelerating agent, and/or with the accelerating agent compatibility
Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material.
It is that term " effective dose " refers to that people and/or animal can be produced function or activity and can be connect by people and/or animal
The amount received." pharmaceutically acceptable carrier " refers to the carrier for Therapeutic Administration, including various excipient and diluent.The art
Language refers to some such medicament carriers：Themselves it is not necessary active ingredient, and does not have undue toxicity after applying.Properly
Carrier be well known to those of ordinary skill in the art.Pharmaceutically acceptable carrier can contain liquid in the composition, such as
Water, brine, buffer solution.In addition, there is likely to be complementary material in these carriers, as filler, lubricant, glidant,
Wetting agent or emulsifying agent, pH buffer substance etc..Lipofectamine can also be contained in the carrier.
In the present invention, can using a variety of methods well known in the art by the accelerating agent or its open gene or
Its pharmaceutical composition delivers medicine to mammal.Including but not limited to：Hypodermic injection, intramuscular injection, for percutaneous administration of, administer locally to,
Implantation, sustained release are given；Preferably, the administering mode is that non-bowel is given.
Preferably, the means of gene therapy can be used to carry out.For example directly the accelerating agent of LOC101930114 can be passed through
The methods of such as injecting delivers medicine to subject；Alternatively, the promotion for carrying LOC101930114 can be adjusted by certain approach
Ceneme (such as expression vector or virus etc.) be delivered on target spot, concrete condition need to regard the accelerating agent type and
Fixed, these are well-known to those skilled in the art.
The effective dose of the accelerating agent of LOC101930114 of the present invention can be with administration pattern and disease to be treated
The order of severity etc. and change.Preferable a effective amount of selection can be come according to various factors true by those of ordinary skill in the art
Fixed (such as passing through clinical test).The factor includes but not limited to：The medicine generation of the accelerating agent of the LOC101930114
Kinetic parameter is such as bioavailability, metabolism, half-life period；The severity of disease that patient to be treated, the body of patient
Heavy, patient immune state, the approach being administered etc..For example, by an urgent demand for the treatment of situation, it can be given once daily and separate several times
Dosage, or dosage is reduced pari passu.
Term " sample " is used with its broadest sense.In a kind of implication, it is intended that including what is obtained from any source
Sample or culture, and biology and environmental samples.Biological specimen is available from animal (including people) and covers liquid, solid, group
Knit and gas.Biological specimen includes blood product, blood plasma, serum etc..It is applicable in however, such sample should not be construed as limitation
In the sample type of the present invention.
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiments are merely to illustrate this
Invent rather than limit the scope of the invention.The experimental method of actual conditions is not specified in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning：Laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment 1 is screened and the relevant gene marker of adenocarcinoma of lung
1st, sample collection
Respectively collect 8 adenocarcinoma of lung cancer beside organisms and pulmonary adenocarcinoma sample.Adenocarcinoma of lung tumor tissues specimen sampling position is
Vital tumor areas, positioned at tumor mass China and foreign countries 1/3 and normal structure junction, excludes the obvious necrosis of tumor center, calcification portion
Point and tumour periphery normal lung tissue；Ai Pang normal lung tissues sample is derived from position more than borderline tumor 5cm, visually observes
Without significant change.The acquirement of above-mentioned all samples passes through the agreement of the committee of organizational ethics.
2nd, the preparation of RNA sample (utilizes E.Z.N.A.Kit is operated)
Liquid nitrogen is imported in mortar, takes the tissue of above-mentioned acquisition to be put into mortar, simultaneously grind into powder is shredded in liquid nitrogen,
Put into after shredding in liquid nitrogen and be ground to powdered, then continued in glass homogenizer；Tissue homogenate adds in glass homogenizer
Enter Trizol reagents, in tissue abrasion on ice.Tissue homogenate after homogenate is transferred in the EP pipes of no RNase, is stood at room temperature
5min.According to the specification extraction separation RNA in kit.It is specific as follows：
1) separation of RNA：
0.2m1 chloroforms are added in EP pipes, cover tightly EP tube covers, 15s is acutely vibrated manually, it is fully mixed.At room temperature
Hatch 5min.Then 15min is centrifuged with 14000g at 4 DEG C.Sample is divided into three layers after centrifugation, and RNA is present in upper strata aqueous phase.
2) RNA precipitate
450 μ l are mutually taken to move in the EP pipes of new no RNase in the water separated, according to 1:It is different that 1 ratio adds 450 μ l
Propyl alcohol, hatches 10min at room temperature after mixing of turning upside down, 4 DEG C of 14000g centrifuge 10min.
3) RNA is eluted
Carefully remove supernatant after centrifugation, add 1ml75% ethanol (destroy the enzyme treatment, matching while using and in precooling on ice) and rinse
RNA, subsequent 4 DEG C of 7500g centrifuge 5min.
4) RNA is redissolved
The careful supernatant removed after washing, opens EP pipe tube covers in superclean bench, places RNA samples at room temperature
5-10min, dries.Add and handle water 20-50 μ l without RNase, water-bath 10min in 55-60 DEG C of water bath.
5) quality analysis of RNA sample
Spectrophotometer detects：
NanoDrop1000 spectrophotometers detect RNA sample, the sample requirement of RNA-seq sequencings：OD260/OD280 is
1.8-2.2。
Agarose gel electrophoresis detects：
By the RNA of said extracted into row agarose gel electrophoresis, Agilent Technologies
2100Bioanalyzer detects RNA sample quality, and observation 28S rRNA and 18S rRNA master tapes are obvious, complete without degraded, RNA
Sex index is qualified, concentration reaches requirement, can be used for the lncRNA express spectras and screening experiment of chip.
3rd, reverse transcription and mark
With Low RNA Input Linear Amplification Kit by mRNA reverse transcriptions into cDNA, while use Cy3
Labelling experiment group and control group respectively.
4th, hybridize
Genetic chip uses Kang Cheng biology-Human lncRNA Array, is carried out by the step of chip operation instructions miscellaneous
Hand over.
5th, data analysis
Chip results are analyzed using Agilent GeneSpring softwares, screening expression quantity has significant difference
(standard differs more than 2 times, and p with the expression quantity by cancer for the lncRNA in cancer<0.05) lncRNA.
6th, result
The results show that expression quantity of the LOC101930114 in pulmonary adenocarcinoma is substantially less than the expression in cancer beside organism
Amount.
The differential expression of 2 QPCR sequence verification LOC101930114 genes of embodiment
1st, large sample QPCR verifications are carried out to LOC101930114 gene differential expressions.Received according to the sample in embodiment 1
Mode set selects adenocarcinoma of lung cancer beside organism and each 50 of pulmonary adenocarcinoma.
2nd, RNA extraction steps are as described in Example 1.
3rd, reverse transcription
1) reaction system：
1 μ l of RNA templates, 1 μ l of random primer, distilled water add to 12 μ l, mix, slow-speed of revolution centrifugation, 65 DEG C of 5min, Ran Houfang
In cooled on ice.
Continuation adds following ingredients in 12 μ l reaction solutions：
5 × reaction buffer, 4 μ l, 1 μ l, 10mM dNTP mixed liquors of RNase inhibitor (20U/ μ l), 2 μ l, AMV reverse transcriptions
1 μ l of enzyme (200U/ μ l)；Fully mix and carry out centrifugal treating；
2) reverse transcription reaction condition
25 DEG C of 5min, 42 DEG C of 60min, 70 DEG C of 5min.
3) polymerase chain reaction
Design of primers：
According to LOC101930114 genes in Genebank and the sequence design QPCR amplimers of GAPDH genes, by winning
Mai De biotech firms synthesize.Specific primer sequence is as follows：
LOC101930114 genes：
Forward primer is 5 '-GGAGATGATGGAGAGTAT-3 ' (SEQ ID NO.2)；
Reverse primer is 5 '-CCACTTATTCCACACTTC-3 ' (SEQ ID NO.3).
GAPDH genes：
Forward primer is 5 '-AATCCCATCACCATCTTCCAG-3 ' (SEQ ID NO.4)；
Reverse primer is 5 '-GAGCCCCAGCCTTCTCCAT-3 ' (SEQ ID NO.5).
Prepare PCR reaction systems：
2 × qPCR mixed liquors, 12.5 μ l, 2.0 μ l of gene primer, reverse transcription product 2.5 μ l, ddH2O 8.0μl。
PCR reaction conditions：95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 60s) × 40 circulations, 60 DEG C of 5min extensions.75
DEG C to 95 DEG C, heat up 1 DEG C per 20s, draw solubility curve.Using SYBR Green as fluorescent marker, in Light Cycler
PCR reactions are carried out on fluorescence quantitative PCR instrument, purpose band are determined by melt curve analysis analysis and electrophoresis, Δ Δ CT methods carry out phase
To quantitative.
5th, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS18.0 statistical softwares, the paired comparisons of cancer and cancer beside organism are examined using t, it is believed that work as P<
There is statistical significance when 0.05.
6th, result
The results are shown in Figure 1, compared with adenocarcinoma of lung cancer beside organism, under LOC101930114 is expressed in pulmonary adenocarcinoma
Adjust, difference has statistical significance (P<0.05) it is, consistent with chip testing result.
3 LOC101930114 of embodiment is overexpressed
1st, cell culture
Human A459 lung cancer cell line, with the RPMI1640 culture mediums containing 10% hyclone and 1%P/S 37 DEG C, 5%
CO2, relative humidity be 90% incubator in cultivate.Change within 2-3 days liquid 1 time, it is conventional using 0.25% trypsase containing EDTA
Had digestive transfer culture.
Cell in blake bottle is digested with pancreatin and is seeded in 6 orifice plates, guarantee cell number is 2-8 × 105A/
Hole, adds cell culture medium.Overnight, second day observation cell density, cell density can be transfected for more than 70%.
2nd, the structure of gene overexpression carrier
According to the synthesis of the cDNA sequence (NR_134509.1, as shown in SEQ ID NO.1) of LOC101930114 special
PCR amplification primer, two restriction enzyme sites of HindIII and XhoI are added in 5 ' end primers and 3 ' end primers respectively.With lung
The cDNA that adenocarcinoma patients' blood extracts and reverse transcription obtains is as amplification template, and above-mentioned cDNA sequence is through restriction enzyme
It is inserted into after HindIII and XhoI double digestions in the eukaryotic expression vector pcDNA3.1 through HindIII and XhoI double digestions,
The recombinant vector pcDNA3.1-1 that connection obtains is used for subsequent experimental.
3rd, transfect
Lung adenocarcinoma cell is divided into 3 groups, is respectively control group (A549), blank control group (transfection pcDNA3.1-NC), reality
Test group (transfection pcDNA3.1-1).The transfection of carrier, the finger of specific transfection method to specifications are carried out using liposome 2000
Show progress.The transfection concentrations of pcDNA3.1 empty carriers and pcDNA3.1-1 are 0.5 μ g/ml.
4th, QPCR detects the transcriptional level of LOC101930114 genes
The extraction of 4.1 cell total rnas
1) cell culture fluid in 6 orifice plates is outwelled, is rinsed twice with PBS, each hole adds 1ml Trizol reagents, room temperature
Place 5min.
2) 0.2m1 chloroforms are added, acutely shake 15s, 4 DEG C, 12000g centrifuges 15min.
3) water is mutually transferred in new pipe, adds 4.5m1 isopropanols, and room temperature places 10min；4 DEG C, 10000g centrifugations 10min.
4) liquid is outwelled, EP tube walls are washed with 75% ethanol of lml.4 DEG C, 7500g, centrifuge 5min.
5) 75% ethanol after cleaning is outwelled, room temperature hangs 5-10min.
6) DEPC water of 25 μ 1 without RNase, -70 DEG C of preservations are added.
4.2 reverse transcription steps are the same as embodiment 2.
4.3 QPCR amplification steps are the same as embodiment 2.
5th, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis, the difference between LOC101930114 gene overexpressions group and control group are carried out using SPSS18.0 statistical softwares
It is different to be examined using t, it is believed that to work as P<There is statistical significance when 0.05.
6th, result
As a result such as Fig. 2 is shown, with non-transfection group compared with transfecting empty plasmid group, transfects and table is crossed in LOC101930114 groups
Up to LOC101930114, difference has statistical significance (P<0.05).
The influence of 4 LOC101930114 gene pairs lung adenocarcinoma cell of embodiment propagation
Testing detection LOC101930114 gene pairs lung adenocarcinoma cells multiplication capacity using CCK-8 influences.
1st, cell culture and transfection procedure are the same as embodiment 3.
2nd, cell to be taken out in second day, micro- Microscopic observation cell growth status, 1ml/ holes add the pancreatin containing EDTA, into
Row cell dissociation, waits to remove pancreatin after the completion of digesting, and adding cell culture medium and mixing makes cell suspend, and then carries out cytometer
Number.
3rd, concentration of cell suspension is diluted to 15000/ml, afterwards toward being inoculated with 96 orifice plates, cell is added per hole
200 μ 1 of suspension, cell are controlled at 3000 or so, are inoculated with 8 multiple holes.PcDNA3.1-1 experimental groups and pcDNA3.1-NC are set
Control group.4 piece of 96 orifice plate is spread altogether is respectively used to 24h, 48h, 72h, 96h4 detection time points.
4th, after 24h, first piece of 96 orifice plate is taken out, the CCK-8 detection liquid of 10 μ 1 is added in every hole, 96 orifice plates are continued to put
Enter and 4h or so is incubated in cell incubator, detect absorbance of each hole at 450nm wavelength with microplate reader and record data.
5th, the operation in 4 is respectively repeated steps after 48h, 72h, 96h, finally counts the absorbance at each time point,
Make growth curve chart.
6th, statistical analysis
Experiment is all completed according to being repeated 3 times, and statistical analysis is carried out using SPSS18.0 statistical softwares, both it
Between difference using t examine, it is believed that work as P<There is statistical significance when 0.05.
7th, result
The results are shown in Figure 3, and compared with the control, after pcDNA3.1-1 is transfected, the propagation of cell is substantially subject to experimental group
Suppress, difference has a statistical significance (P<0.05) illustrate that LOC101930114 has the function that to suppress cell Proliferation.
The influence of 5 LOC101930114 gene pairs Apoptosis of Lung Adenocarcinoma Cell of embodiment
Use the influence of flow cytomery LOC101930114 gene pairs Apoptosis.
1st, cell culture step is the same as embodiment 3.
2nd, cell transfecting step is the same as embodiment 3.
3rd, step
1) 10 × sample-loading buffers of 3m1 27m1 distilled water is diluted.
2) collection of cellular samples and cleaned with the PB S of precooling.
3) cell is added into 1 × sample-loading buffers of lml, 300g centrifugation 10min, suction out buffer solution.
4) 1 × sample-loading buffers of lml are added again, and cell concentration in cell suspension is adjusted to 1 × 106A/ml.
5) cell suspension is taken out into 100 μ 1, added in EP pipes.
6) the Annexin V FITC of 5 μ l are added in EP pipes, mixes the liquid in EP pipes, lucifuge is incubated at room temperature
10min。
7) 5 μ 1PI dye liquors are added into EP pipes, at room temperature lucifuge 5min.
8) PBS solution of 500 μ l is added in EP pipes, is gently mixed, flow cytometer is detected in 1h.
3rd, statistical method
Experiment is all completed according to being repeated 3 times, and result data is represented in a manner of mean+SD,
Statistical analysis is carried out using SPSS13.0 statistical softwares, the t of difference use between the two is examined, it is believed that works as P<When 0.05
With statistical significance.
4th, result：
The results are shown in Figure 4, and for experimental group compared with control group, apoptosis rate has significant change (P<0.05), should
As a result illustrate, LOC101930114 has obvious influence to the apoptosis of lung adenocarcinoma cell.
6 cell migration of embodiment and Matrigel
1st, prepared by Transwell cells
Matrigel is ice bath melted under aseptic condition, carries out 20 times of dilutions with PBS, is layered on the volume in 50 μ l/ holes
On the polycarbonate membrane of Transwell cells.37 DEG C of placement 4h, take out after Matrigel glue aggregates into gel, gently suction out
Upper strata separates out liquid.The serum-free medium containing BSA that 50 μ l are added in per hole carries out basilar memebrane hydration process, 37 DEG C of placements
30min。
2nd, cell suspension is configured
Cell removes serum starvation processing 12-24h, carries out digestion process to cell, is centrifuged after terminating digestion, in removal
Layer nutrient solution.Sedimentation cell is cleaned with PBS, the serum free medium containing BSA is added and it is resuspended.Adjustment is thin
The density of born of the same parents is to 5 × l05A/ml.
3rd, cell inoculation
200 μ 1 (migration experiment is 100 μ 1, and Matrigel is 200 μ 1) of cell suspension is taken to be added to Transwell cells
In.Room adds 1640 culture mediums of 500 μ 1 containing FBS under 24 orifice plates.Cell is put into cell incubator and cultivates 24h.
4th, dye
Cell is dyed after culture using DAPI.Small ventricular cell is first rinsed 2 times with PBS, is put into DAPI working solutions
Middle room temperature dyes 5-20min.Rinsed 2 times with PBS, be put into fluorescence microscopy Microscopic observation and count.
5th, result
The results are shown in Figure 5, after lung adenocarcinoma cell transfected plasmids, compared with control group, the migration of experimental group and invades
Attack ability to be decreased obviously, as a result illustrate that LOC101930114 can suppress the migration and invasion and attack of adenocarcinoma of lung.
The explanation of above-described embodiment is only intended to understand the method and its core concept of the present invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, some improvement can also be carried out to the present invention
And modification, these are improved and modification will be also fallen into the protection domain of the claims in the present invention.
SEQUENCE LISTING
<110>The 4th hospital of Hebei Medical University
<120>Applications of the lncRNA as biomarker in adenocarcinoma of lung diagnosis and treatment
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 456
<212> DNA
<213>People source
<400> 1
cacactctgc tcaccaccct gtgggccttg tcccaggact catcccttct cttctggagc 60
catttccaat tctgcccaaa aggacagcct gaagtgctgg accctgggta cctggctgaa 120
gactttggac tttaacctgg agatgatgga gagtatgcag gataaagtcc ataccaaatc 180
caacgcctag caaatggaga acaagccaaa actaacaaga taaaatgtag caggaaaaag 240
tgcaaagagc tgaagtgtgg aataagtggg agaggtctgc catgccctat ctgaccttgc 300
tgccatgatc tttaaggctg tgtgtcctgg ttggtgcagc tacaagatgg cagaaatgcc 360
taagtgatgg tatggaacag aggatccctc ttctcaccat tgtcttgcat gtttccttgt 420
gtgaaccaca aataaacttt tgttatgtta agctgc 456
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
ggagatgatg gagagtat 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence
<400> 3
ccacttattc cacacttc 18
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
aatcccatca ccatcttcca g 21
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<400> 5
gagccccagc cttctccat 19
Claims (5)
1. application of the reagent of LOC101930114 gene expressions in the product for preparing diagnosis adenocarcinoma of lung is detected, it is described
The sequence of LOC101930114 genes is as shown in SEQ ID NO.1.
2. application according to claim 1, it is characterised in that the product includes chip, preparation or kit.
3. purposes of the LOC101930114 genes in human lung adenocarcinoma medicine is screened, the LOC101930114 genes
Sequence is as shown in SEQ ID NO.1.
4. application of the LOC101930114 genes in the pharmaceutical composition for preparing treatment adenocarcinoma of lung, the LOC101930114
The sequence of gene is as shown in SEQ ID NO.1, it is characterised in that described pharmaceutical composition includes the rush of LOC101930114 genes
Into agent and/or the accelerating agent of its expression product.
5. application according to claim 4, it is characterised in that described pharmaceutical composition further includes and the accelerating agent compatibility
Other medicine classes and pharmaceutically acceptable carrier and/or auxiliary material.
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| CN109055556A (en) * | 2018-08-27 | 2018-12-21 | 中山大学 | A kind of lncRNA detection kit and its application for diagnosing transfer |
| CN111254146B (en) * | 2020-03-13 | 2021-10-26 | 四川诺沃赛尔生物科技有限公司 | Application of LINC01331 gene inhibitor in preparation of medicine for treating lung cancer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282385A (en) * | 2016-10-14 | 2017-01-04 | 天津医科大学肿瘤医院 | Long-chain non-coding RNA XLOC_000090 qualification in pulmonary carcinoma and purposes |
| CN106319043A (en) * | 2016-08-18 | 2017-01-11 | 中南大学 | Application method of long non-coding RNA LINC 01420 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319043A (en) * | 2016-08-18 | 2017-01-11 | 中南大学 | Application method of long non-coding RNA LINC 01420 |
| CN106282385A (en) * | 2016-10-14 | 2017-01-04 | 天津医科大学肿瘤医院 | Long-chain non-coding RNA XLOC_000090 qualification in pulmonary carcinoma and purposes |
Non-Patent Citations (3)
| Title |
|---|
| Accession ID:NR_134509.1 Homo sapiens uncharacterized LOC101930114 (LOC101930114)，transcript variant 1，long non-coding RNA;Bonaldo MF等;《GenBank Database》;20161009;ORIGIN部分 * |
| Analysis of lncRNA expression profiles in non-small cell lung cancers(NSCLC)and their clinical subtypes;Jingcheng Yang等;《Lung Cancer》;20140831;第85卷(第2期);第110-115页 * |
| lncRNA与非小细胞肺癌;张磊等;《中国肿瘤外科杂志》;20150630;第7卷(第3期);第192-195页 * |
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